The present invention concerns an improved method and kit for the diagnosis of Chlamydia trachomatis (C. trachomatis) in humans. More specifically, the present invention concerns a new mixture of peptides derived from the major outer membrane protein (MOMP), which together are capable of specifically reacting only with antibodies specific to any one of the serovars of C. trachomatis, and hence said mixture of peptides is particularly useful for identifying C. trachomatis infections in humans by binding specifically to antibodies, if present, in a body fluid sample obtained from an individual being tested.
Chlamydia is a gram negative obligate intracellular bacteria that causes acute and chronic disease in mammalian and avian species. The genus Chlamydia is comprised of four species: C. trachomatis, C. pneumonziae, C. precorum and C. psittaci. 
The C. trachornatis species is divided into 15 serovars. Serovars A, B, Ba and C are agents of trachoma, a leading cause of preventable blindness, endemic in the Third World. Serovars L1-L3 are the agents of lymphogranuloma venereum. Serovars D-K are a common cause of sexually transmitted genital infection worldwide: cervitis, endometritis/salpingitis in females and uretritis in both males and females. Endometritis/salpingitis can lead to agglutination of salpinx, with a higher risk of extra-uterine pregnancy and infertility. The genital infection can cause acute infection and persistent infection occasionally without any clinical sign. Generally, these infections are treatable, once they are diagnosed; however, without any treatment, the infections can progress to severe chronic inflammation leading to infertility, ectopic pregnancy, induced abortion or pre-term child delivery. Moreover, the infants of infected mothers can themselves be infected during birth, leading to conjunctivitis or pneumonia.
Serological testing, i.e., the testing for the presence (or not) of anti-C. trachomatis antibodies in an individual, is now an established approach in many countries, and has been shown to provide a comprehensive answer for the detection of C. trachomatis infection. In suspected deep-seated infections, body fluid sampling reduces the necessity for invasive procedures which are required for direct antigen detection. In cases of lower urogenital infections, collection limitations such as effectiveness of scrape sampling procedure, specimen handling and transportation difficulties have to be weighed. Above all, there however still remains the problematic issue that most Chlamydial infections are asymptomatic. Therefore, an infection may persist for a long time, ascend the upper genital tract, causing deep and chronic infections, and increase the probability of false negative results in procedures designed for direct antigen detection, i.e., sampling of lower genital tract tissue with anti-C. trachomatis antibodies, or other suitable procedures, to directly detect the presence of the major C. trachomatis antigens such as the major outer membrane protein (MOMP) indicative of Chlamydial infection.
Serological testing for Chlamydia trachomatis, through the detection of various specific antibodies, is today an effective and highly accepted optional detection procedure. New and refined technologies apply the immuno markers IgM, IgA and IgG to characterize the presence and stage of infection.
The detection of specific IgM antibodies is indicative of acute Chlamydial infections. The absence does not, however, preclude the presence of on-going infection, however, especially in recurrent and chronic cases. The detection of specific IgA antibodies is now accepted as indicative of active Chlamydial infection and has been shown to be an important marker because of the shorter lifetime of IgA antibodies which persist only as long as antigenic stimulation exists. IgA antibody detection is, moreover, suitable for post-therapy follow-up. IgG antibody detection is a marker for Chlamydial-positive immune-response, either for current, chronic or past infections.
There exists a high level of serological cross-reactions between the three different species of Chlamydia. Most of the serological diagnostic assays for Chlamydia use either purified elementary bodies (microimmunofluorescence, MIF and ELISA tests), lipopolysaccharide, LPS or purified major outer membrane protein, MOMP (ELISA tests) as antigens. Genus specific epitopes are present in all the above antigens, therefore, low species specificity is observed. Moreover, a large proportion of the worldwide human population has been exposed to C. pneumoniae (with no clinical signs) and the prevalence of anti-Chlamydia antibodies is very high. Therefore, the differentiation between C. pneumoniae and C. trachomatis specific antibodies using conventional serological screening tests (MIF, ELISA, EIA, etc.) is not very effective.
A number of publications have been made in which there have been disclosed the isolation, purification and characterization of the major C. trachomatis outer membrane complex proteins, including the above-noted MOMP, and the use thereof for the purposes of generating anti-C. trachomatis antibodies and in immunoassays to detect the presence of anti-C. trachomatis antibodies in samples obtained from individuals suspected of being infected by C. trachomatis. For example, in U.S. Pat. No. 5,318,892 and its corresponding EP 0 456 524, there is disclosed the use of C. trachomatis outer membrane complex consisting of at least three polypeptides, including MOMP, for assaying anti-C. trachomatis antibodies. In U.S. Pat. No. 4,427,782, there is described the isolation and characterization of the C. trachomatis MOMP and its use in immunoassays. However, in the aforesaid published patents, there is not provided any nucleotide or amino acid sequences of the MOMP polypeptide, nor is there disclosed the possibility of using peptides derived from MOMP for the purposes of immunization against C. trachomatis disease or for use in immunoassays to detect anti-C. trachonzatis antibodies, indicative of infection by C. trachoniatis. It is well known in the art that large proteins such as MOMP are less effective than smaller antigenic peptides derived therefrom, both for the purposes of immunization against C. trachomatis infection and for use in immunoassays to detect anti-C. trachomatis antibodies. Further, the complete MOMP protein obtained from C. trachomatis also has a number of epitopes which are common to the MOMP protein obtained from the other above-noted species of Chlamydia, with the result that using the complete protein in immunoassays to specifically detect anti-C. trachomatis antibodies is not reliable because of the possibility of cross-reactivity between the various Chlamydia species, with the result that when positive results are obtained, it is not readily possible to determine whether the antibodies are specific to C. trachomatis or to the other Chlamydia species. Hence, in respect of C. trachomatis, false-positive results may be obtained and may even lead to an incorrect diagnosis of the agent causing the infection and subsequently to an incorrect mode of treatment of the infection. Moreover, large proteins like MOMP are often also difficult to produce and purify, especially when high levels of purity are imperative when such proteins are to be used in diagnostic kits, and such large proteins are often difficult to incorporate into a kit, for example, a kit based on ELISA in which the antigen is usually immobilized on a solid support.
In other publications, there has been described the cloning and sequencing of MOMP derived from C. trachomatis, the production of recombinant C. trachomatis MOMP polypeptide and its use for raising anti-MOMP antibodies and its use in immunoassays to detect anti-MOMP antibodies specific to C. trachomatis. The aforesaid cloning and sequencing of MOMP has been described, for example, in EP 0 192 033, in which publication there is also described the production of various monoclonal antibodies, each recognizing a different specific epitope in MOMP. However, for the reasons noted above, even such a recombinant MOMP polypeptide still suffers from the drawbacks of having possible cross-reactivity with the MOMP proteins from the other Chlamydia species as regards recognition of anti-MOMP antibodies. Further, even recombinantly produced MOMP requires a significant input of resources and time, both for the production thereof and for the purification thereof, before it is suitable for use in diagnostic assays or as a vaccine.
A number of publications have described various peptides derived from MOMP which are useful as vaccines and also in immunoassays to detect anti-MOMP antibodies. To obtain such peptides has been possible following the elucidation of the full sequence of MOMP and its analysis with respect to variation of the sequence within the different serovars. For example, there has been described an analysis of the various domains of the C. trachomatis MOMP protein, namely, the fact that it is divided into four constant (CDI-IV) and four variable (VDI-IV) domains (Yuan et al. Infect. Immun. 57, p. 1040-1049, 1989). Based on such and similar data, peptides were designed that generally have sequences corresponding to those parts or regions of MOMP which were most variable, and therefore expected to also be most antigenic. This means that such peptides essentially represent epitopes of MOMP to which it is readily possible to raise antibodies and hence such peptides may be used as vaccines, or may be readily used in diagnostic assays to detect the presence of anti-MOMP antibodies. For example, in EP 0 348 725, there is described an epitope-specific peptide which is a common epitope of C. trachomatis that is useful for the production of specific antibodies to C. trachomatis MOMP and which peptide has a sequence derived from the MOMP sequence between amino acid residues 262 and 320. Further, in WO 94/06827, there is disclosed a synthetic peptide comprising a T-cell helper cell stimulating epitope and a B-cell neutralizing antibody stimulating epitope derived from the C. trachomatis MOMP. This synthetic peptide is useful in immunoassays to detect anti-C. trachomatis MOMP antibodies or as a vaccine. However, while the aforesaid peptides represent an important improvement over use of the complete MOMP polypeptide as regards their usefulness in diagnostic assays to detect C. trachomatis antibodies or their usefulness as vaccines, such peptides still suffer from one major drawback, namely, these peptides do not always define an epitope that is common to all of the above-noted serovars of C. trachomatis. As a result, when such peptides are used to detect C. trachomatis antibodies, they are not capable in all instances of detecting antibodies against all of the various serovars of C. trachomatis and subsequently false-negative results may be obtained in such diagnostic assays using such peptides, namely, an individual may have been infected with a certain serovar of C. trachomatis common to the area where such individual lives, but the peptide used in the assay may be one which does not fully correspond to the specific epitope of the specific serovar with which the individual has been infected with the result that antibodies raised in this individual will not react fully or will not react at all with the peptides in the diagnostic kit, resulting in the obtention of a false-negative result.
Hence, heretofore there has not been disclosed a C. trachomatis MOMP-specific peptide or mixture of peptides which are specific for all the different serovars of C. trachomatis and which may be used in diagnostic assays to detect anti-MOMP antibodies raised against any of the C. trachomatis serovars, or which may be used as vaccines to immunize people effectively against all of the various serovars of C. trachomatis. As mentioned above, C. trachomatis infections occur in a very large number of people worldwide and hence there has been a long-felt need to overcome the above-mentioned drawbacks of the prior art and to provide a peptide or mixture of peptides which may be used in diagnostic assays to detect anti-C. trachomatis MOMP antibodies raised in individuals following infection by any of the serovars of C. trachomatis. Likewise, it has been a long-felt need to provide a peptide or mixture of peptides, which when administered to an individual will be capable of eliciting anti-MOMP antibodies specific to any of the serovars of C. trachomatis and in this way provide for an effective vaccine against C. trachomatis infection.
Accordingly, it is an aim of the present invention to provide a mixture of peptides derived from specific regions within the MOMP protein, which together are capable of interacting with antibodies against MOMP from any of the serovars of C. trachomatis. Such a mixture of peptides is particularly useful for diagnostic assays to diagnose the presence of anti-MOMP antibodies specific to C. trachomatis obtained from the body fluids of an individual and being specific for all of the serovars of C. trachomatis, such diagnostic assays should essentially provide no false-negative results. Likewise, as the mixture of peptides is chosen, in accordance with the present invention, to be specific only to the MOMP of C. trachomatis serovars and not to MOMP from any other Chlamydia species, use of the peptides in such diagnostic assays should also provide these diagnostic assays as having essentially no false-positive results.
Another aim of the present invention is to provide for a diagnostic kit for diagnosing the presence in a body fluid of anti-C. trachomatis MOMP antibodies of any of the C. trachomatis serovars, this diagnostic kit, using such a mixture of peptides as noted above, providing for a highly reliable diagnostic test of test samples being inherently essentially incapable of yielding false-positive or false-negative results. This diagnostic kit is also highly specific only to anti-C. trachomatis MOMP antibodies and does not cross-react with anti-MOMP antibodies specific to the other species of Chlamydia.
It is another object of the present invention to provide for a method of diagnosing Chlamydia or C. trachomatis infection by detection of anti-C. trachomatis antibodies in the body fluid of an individual using as test reagent the above-mentioned mixture of peptides.
A still further object of the present invention is to use the above mixture of peptides as a vaccine to immunize an individual against C. trachomatis infection by any of the serovars of C. trachomatis. 
Other objects and aspects of the present invention will arise from the following detailed disclosure of the invention.
The term xe2x80x9cbody fluidxe2x80x9d, as used herein, comprises fluids sampled from within the body, such as blood or lymph, local secretions, such as tears, semen, urine, sweat, sputum etc., samples obtained by washing (e.g. bronchiolar lavage) or swabbing, including cervical smears, and the like.
The term xe2x80x9cpeptidexe2x80x9d, as used herein, comprises peptides obtained by chemical synthesis, or by cleavage, either by chemical means or by using proteolytic enzymes, of a larger peptide or protein.
In the following detailed disclosure of the invention, there will be provided, amongst other details, the amino acid sequences of the various peptides constituting the mixture of peptides in accordance with the present invention. These sequences will be written in the form of the single letter amino acid code. For the purposes of clarity, the following is the key to this single letter code:
Further, as will be appreciated by all skilled artisans, the sequences of the peptides in accordance with the present invention have been derived from the known and published C. trachomatis MOMP amino acid sequence which has been elucidated for all the various C. trachomatis serovars. For example, the above-noted EP 0 192 033 provides the full sequence of MOMP, the above EP 0 348 725 provides the sequences of various MOMP-derived peptides and the above WO 94/06827 provides the sequences of MOMP-derived peptides from the various C. trachomatis serovars. In these publications, reference is also made to a number of other publications in which the full sequences of MOMP from various serovars has been published. Hence, all of the aforesaid publications and relevant publications indicated therein are included in the present specification in their entirety as concerns the known sequences of MOMP from all the C. trachomatis serovars.
The present invention is based on the finding that by modifying certain specific peptides derived from the second and fourth variable domains of the immunodominant MOMP protein of C. trachomatis and preparing a mixture of such peptides it is possible to obtain a mixture of such peptides that is highly specific to anti-MOMP antibodies specific to the MOMP of C. trachoniatis and which has no cross-reactivity with anti-MOMP antibodies specific to the MOMP of other Chlamydia species such as C. pneumoniae or C. psittaci. Further, it has also been found in accordance with the present invention that by using such a mixture of peptides in a kit for the diagnosis of C. trachomatis disease, there is provided a kit having very high sensitivity and very high specificity towards IgA or IgG antibodies specific for C. trachomatis MOMP when present in a test body fluid sample assayed with this kit.
Accordingly, the present invention provides a mixture of peptides derived from the variable domains of the Chlamydia trachomatis (C. trachomatis ) immunodominant major outer membrane protein (MOMP), said mixture characterized by having specificity only to C. trachomatis anti-MOMP antibodies and being non-cross reactive with anti-MOMP antibodies of other Chlamydia species and said mixture also characterized by having specificity to anti-MOMP antibodies from all serovars of C. trachomatis, said mixture of peptides comprising about 4 peptides being selected from the group of peptides consisting of:
(a) SEQ ID NO. 1, herein also designated peptide 4A, having the amino acid sequence:
IFDTTLNPTIAGAGDVK;
(b) SEQ ID NO. 2, herein also designated peptide 4B having the amino acid sequence:
VDITTLNPTIAGCGSVAK;
(c) SEQ. ID NO. 3, herein also designated peptide 4C, having the amino acid sequence:
CVFDVTTLNPTIAGAGDVK;
(d) SEQ. ID NO. 4, herein also designated peptide 4D, having the amino acid sequence:
LAEAILDVTTLNPTITGKAVVSK;
wherein all of said peptides (a)-(d) are derived from the fourth variable domain (VDIV) of said MOMP;
(e) SEQ. ID NO. 5, herein also designated peptide C.t2A having the amino acid sequence:
CDNENQSTVKTNSVPNMSLDQSK, wherein said peptide (e) is derived from the second variable domain (VDII) of said MOMP;
(f) SEQ. ID NO. 6, herein also designated as C.t VDI, having the amino acid sequence:
VAGLENDPTTNVARA;
(g) SEQ. ID NO. 7, herein also designated as C.t VDII, having the amino acid sequence:
DNENNATVSDSKLVPNHMSDQS;
(h) SEQ. ID NO. 8, herein also designated as C.t VDIV, having the amino acid sequence:
LDVTTNATIAGKGTVV;
(i) analogs of any one of peptides (a)-(h), wherein said analogs have essentially the same biological activity as said peptides (a)-(h), said analogs having between about 9 to about 35 amino acid residues and differing from said peptides (a)-(h) by having one or more additions, deletions or substitutions, or by being combinations of an N-terminal part of one peptide selected from (a) to (d) or (h) with a C-terminal part of another peptide selected from (a) to (d) or (h), such that the complete sequence will be homologous to the VDIV sequence, or by being combinations of an N terminal part of one peptide selected from (e) or (g) with a C-terminal part of another peptide selected from (e) or (g), such that the complete sequence will be homologous to the VDII sequence.
Preferred mixtures in accordance with the invention are:
A mixture of peptides, wherein the peptides are analogs of peptides (a) to (h), and wherein all of said analogs are chosen to correspond to a MOMP VD sequence of one or more of the groups of serovars.
A mixture of peptides as defined above, characterized in that the mixture has specificity to a single serovar or group of serovars of C. trachomatis only.
A mixture of peptides, said mixture characterized by having specificity to anti-MOMP antibodies from all serovars of C. trachomatis. 
Preferred mixtures of peptides in accordance with the present invention having specificity to anti-MOMP antibodies from all serovars of C. trachomatis are:
A mixture of peptides noted above being a mixture of 4 peptides (a), (b), (c) and (d), or a mixture of one or more of said peptides (a)-(d) with one or more analogs of (a)-(d), wherein, when said mixture contains 4 peptides selected from any of the possible combinations of (a) or analog of (a)+(b) or analog of (b)+(c) or analog of (c)+(d) or analog of (d);
A mixture of peptides as noted above being a mixture of 4 peptides (a), (b), (c) and (e), or a mixture of one or more of said peptides (a)-(c) and (e) with one or more analogs of (a)-(c) and (e), wherein, when said peptides and said analogs constitute said mixture, the mixture contains 4 peptides selected from any of the possible combinations of (a) or analog of (a)+(b) or analog of (b)+(c) or analog of (c)+(e) or analog of (e);
A mixture of peptides as noted above being a mixture of said peptides (a), (b), (c) and (d), wherein said peptides are present in said mixture in equal amounts;
A mixture of peptides as noted above being a mixture of said peptides (a), (b), (c) and (e), wherein said peptides are present in said mixture in equal amounts.
The present invention also provides the said peptides conjugated directly or indirectly (e.g., via beads and/or linkers) to a detectable marker, such as, for example, a radioactive label, an enzyme, avidin or a derivative thereof, biotin, digoxygenin, gold, ligands, haptens, metals or metal compounds having magnetic properties, and the like.
The present invention also provides a kit for the diagnosis of C. trachomatis infection comprising any of the above mixtures of peptides and manufacturer""s instructions for use of said kit.
Preferred kits according to the invention are selected from RIA, EIA, ELISA, Immuno competition assays, lateral chromatography assays, and the SeroRapid(copyright) assay.
A particularly preferred kit according to the present invention is a kit carrying out an enzyme linked immunoassay (ELISA), and said kit comprises said mixture of peptides immobilized on a solid support and the reagents for performing said ELISA.
Likewise, the present invention also provides for the use of any of the above mixtures of peptides for the preparation of a diagnostic kit for the detection of C. trachomatis infection.
Furthermore, the present invention also provides for a method for detecting C. trachomatis infection in an individual comprising:
(a) obtaining a body fluid sample from said individual;
(b) contacting said body fluid sample with any of the above mixtures of peptides under suitable assay conditions; and
(c) determining the extent of reaction between said body fluid sample and said mixture of peptides.
Preferred body fluid samples are serum, saliva, tear fluid, urine, semen, cervical smears, bronchiolar lavage and sputum.
A preferred method of the present invention is a method comprising performing a diagnostic assay of the ELISA type, wherein said mixture of peptides is immobilized on a solid support, and then contacted under suitable ELISA conditions with said body fluid sample and wherein if said body fluid sample contains antibodies specific for C. trachomatis MOMP, there will be a detectable positive reaction with said mixture of peptides.
Preferred body fluids for diagnosis are serum, saliva, urine and tear fluid. Serum is particularly preferred.
Further, the present invention provides for the use of the said peptides for the purpose of vaccination. The mixture of the peptides may be used in order to achieve immunization against all strains of C. trachomatis, while single peptides may be used in order to achieve immunization against groups of C. trachomatis serovars, like e.g., the group consisting of serovars A-C, or D-K, or of the different L serovars.
Other aspects and embodiments of the present invention will be set forth in the following detailed description of the invention or will clearly arise therefrom.